Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
The phylum Apicomplexa includes hundreds of different organisms belonging to the order Eucoccidiorida. The genus is included within the order of true coccidian agents. Of the organisms belonging to this genus, several species are of recognized importance to the chicken industry. These species include Eimeria tenella, E. maxima, E. acervulina, E. necatrix, E. brunetti, E. mivati E. mitis and E. praecox.
Differentiation of species is based on the site of infection within the host and oocyst morphology. To date, biochemical markers have not been used for speciation, although differences have been noted for each of the above species. For Eimeria, the entire life cycle is completed within a single host. The actual stages of the life cycle vary in complexity depending upon the Eimeria species involved. For example, E. necatrix has a complex life cycle pattern. Upon being ingested in contaminated feces, food or water, sporulated oocysts excyst within the digestive tract as a result of the combined action of mechanical shearing and enzymatic hydrolysis of the sporocyst cap. The liberated sporozoites traverse epithelial cells within specific regions of the intestine. Development begins within the Crypt of Lieberkuhn to the level of first generation meronts; the meront is a transitional stage consisting of rounded organisms with a more pronounced nucleus, plus increased energy generating and protein synthesizing capacity. Development of first-generation merozoites follows due to multiple fission of meronts. The release of first-generation merozoites destroys the host cell, and the parasites migrate to infect new host cells undergoing a second asexual cycle. Meronts develop to the level of second-generation merozoites destroying additional epithelial cells as they are released. Further destruction of host cells follows with the liberation of the third-generation merozoites. Second- and third-generation merozoites may infest still another population of host enterocytes to begin the sexual phase. Sexual development commences with the production of microgametes and macrogametes through the process of gametogenesis. Liberated microgametes fertilize macrogametes to form zygotes. Development of immature oocysts is followed by rupture of the host cell. Oocysts, released into the lumen of the gut, are passed through the feces to the environment and mature (sporulate) in the presence of atmospheric oxygen.
The process of parasite development is self-limiting if the host ingests no additional oocysts. However, this proves to be an unrealistic expectation in crowded poultry houses.
Disease due to E. necatrix can result in severe economic losses associated with diminished feed efficiency and pathologic manifestations.
The pathology of coccidiosis due to E. necatrix and some other species is in large part related to the rupture of host cells during the release of merozoites. Tissues are disrupted primarily within the mid-gut subepithelium. Bleeding within the gut is related to rupture of small capillaries servicing the epithelium. It may be difficult to control the progress of disease using coccidiostats, once asexual development is established. Secondary infection often complicates the disease caused by Eimeria. Death can occur within 4-7 days in severely infected birds.
A consistent property of the coccidia is that the sporozoites initiate the infection process within very specific tissue sites (29, 34, 42). The site specificity of infection is a characteristic commonly used for speciation of Eimeria. The asexual stages of E. necatrix show a propensity for invasion of epithelial cells residing within the mid-intestine. Sexual stages develop primarily in the cecal pouches.
Investigation of immunity to coccidiosis has examined the role of humoral immunity, and more specifically of serum antibody. Studies have shown a lack of correlation between serum antibody and resistance to disease (44). However, most available data support the contention that a local response with involvement of the secretory immune system or cell mediated immunity (CMI), or both, are involved in the protective response.
Interference with recognition, penetration and/or attachment of pathogens to host cells has a demonstrated protective effect as shown with viral, bacterial and protozoan models. Genetic deletion of key host cell receptors or pathogen attachment features can prevent the initial colonization process (13, 41). Alternatively, secretory antibodies can interfere with the colonization process by binding to, and consequently masking requisite receptors (23, 54). More than one immunoglobulin class has been reported to have the capacity of interfering with the initial colonization process of Eimeria tenella (10). However, recent reports indicate that only production of secretory IgA has been correlated with natural protective immunity (9, 44). Porter and Davis (10) and others (44) reported that secretory IgA neutralizes the extracellular stages of the parasite either by significantly limiting penetration or so debilitating those organisms which did penetrate as to prevent subsequent development.
It has been estimated that an amount approaching $0.5-1.0 billion is spent annually by producers worldwide to combat disease, or the effort to curb the devastating effect of coccidiosis in chickens (29, 39). Currently, the most widely used means of controlling Eimeria in chickens is through the application of antiprotozoal chemical feed additives. The specific composition varies with the coccidiostat used, and each product affects only certain stages of the coccidian life cycle (29, 38, 43). Disadvantages of using coccidiostats are many, including short-term residual protection in birds, occasional diminished performance, invocation of resistance to the drug in parasites, and to some extent, safety. Products currently remain on the market for only a few years because of the development of drug resistant strains. This adds considerable pressure on the cost of development and continued manufacture of efficacious products (38).
Protection of birds by immunization has met with some success. Investigators have been able to invoke limited protection using preparations of killed organisms (1, 31, 32). A more effective approach for immunization of chickens has been with the use of a live protozoal product - - - e.g. Cocciac.TM. (12). The product, being a multivalent composition containing low doses of viable oocysts, is administered in drinking water to invoke a mild parasitemia in birds. A drawback of this product has been occasional depressed performance of birds during the first weeks following administration. Variables such as excessive dosing or moisture content of bedding have even led to severe outbreaks of coccidiosis. See also, U.S. Pat. No. 3,147,186 (1964) which concerns the use of viable, sporulated oocysts of E. tenella to immunize chickens and U.S. Pat. No. 4,301,148 (1981) which concerns the use of sporozoites of E. tenella for the same purpose.
An alternative means of introducing the live vaccine into broiler houses is by way of the feed. This has been considered in a recent British patent (GB2,008,404A). Prior to mixing with the feed, fully virulent oocysts of E. tenella are encapsulated in a water soluble polysaccharide to protect against desiccation. The oocysts are in sufficient amounts only to induce subclinical infection. Though the immunizing ability was found to be excellent, no development of this method is foreseen due to questionable field acceptability. However, if attenuated strains of all the important coccidia could be developed, the procedure may be more acceptable.
Efforts have indeed been made to develop Eimeria lines of reduced virulence. Some species have been successfully attenuated through chicken embryo passage (14, 27, 30, 48). These strains have diminished ability to cause disease, yet have retained sufficient immunogenicity to invoke immunity. Some problems do, however, remain with the handling of these strains. As examples, the attenuated variants of E. necatrix have a critical passage limit whereby more or less embryo passage can result in loss of immunogenicity or maintenance of the original virulent form. Furthermore, some attenuated organisms revert to the virulent form upon minimal back-passage through chickens (28, 50). Thus, problems associated with maintaining consistent properties in attenuated organisms are apparent.
Attenuation by precocious selection has also been practiced when Eimeria strains cannot be readily passaged through embryonated eggs. In this process, shed oocysts are harvested late in the prepatent period prior to the onset of heavy oocysts shedding (19, 35, 37, 49). Such selection results in cultures having abbreviated life cycles, and a corresponding diminution in virulence properties (19, 35, 37, 49). Though the trait of precocity for E. tenella (20) and E. acervulina (36) has been demonstrated to be genetically stable, not enough information is known about this method to assess its usefulness as a tool in the poultry industry.
There is little information available about the surface antigen composition of avian coccidia. Hybridoma cell lines which secrete monoclonal antibodies directed to antigens on the surface of sporozoites of Eimeria tenella have been reported (59). The antigens were not identified, other than that their molecular weights were between 13 and 150 kilodaltons. Moreover, no biological significance or described efficacy in a vaccine was attributed to the antigens. Previous work in the laboratory of M.H. Wisher suggests the presence of approximately 16 polypeptides identified by surface iodination of excysted sporozoites of E. tenella and having molecular weights form 20,000 to greater than 200,000 (58).
Subunit approaches to vaccine development have proven successful over the past few years. In such approaches, candidate protective antigens are identified and characterized for the purpose of eventual preparation on a large scale. In studying parasite antigens, one research group used monoclonal antibodies to identify a potential protective antigen on the surface of Babesia bovis (60). A B. bovis antigen of 44,000 daltons has been identified, which when purified and injected into experimental animals afforded some level of protection against primary challenge. An immunologically important 30,000 dalton protein of Toxoplasma gondii has also been identified using monoclonal antibodies (22).
Since mid-1981, Danforth and coworkers have published several papers in which they indicate the possibility of producing monoclonal antibodies toward antigens of avian Eimeria species (6, 7, 8). Similarly, Speer, et al. (51, 52) have demonstrated the development of hybridomas against E. tenella and some physiologic properties thereof. Antibody-secreting hybridomas have been selected on the basis of an indirect fluorescent antibody test (7). The patterns of reaction, as observed with ultraviolet microscopy, have varied depending upon the monoclonal antibody used. Patterns have included exclusive reaction with sporozoites only vs reaction with sporozoites and merozoites; staining of the anterior portion of the sporozoite the entire membrane; and staining of distinct internal organelles vs non-descript internal staining (8).
Although the preparation of murine-origin hybridomas producing monoclonal antibodies is commonly practiced by those familiar with the art, there is nothing to suggest that the direct and specific selection of sporozoite-neutralizing hybridomas, against the species E. necatrix will subsequently identify virulence determinants of E. necatrix useful in the development of a subunit vaccine.
This invention concerns the identification, characterization, preparation and use of polypeptide antigens for development of immunity to coccidiosis by Eimeria necatrix and Eimeria tenella.
The antigens are capable of being precisely dispensed in terms of direct antigenic content and cannot cause disease thus avoiding vaccine strain-related outbreaks and reversions or changes in immunologic properties.